In the present research application, experimental, bioinformatics and computational techniques are utilized to probe important amino acid interactions at the dimer interface of human glutathione synthetase (hGS) in light of the negative cooperativity of hGS. The mechanism whereby the two active sites of hGS communicate across the dimer interface, despite their ca. 50 E separation will be investigated. The hypothesis that amino acids with strong interactions across the dimer interface play a critical role in hGS negative cooperativity will be tested. Aim 1: The most important residues that facilitate the subunit:subunit communication as well as the types of interactions will be identified using computational modeling techniques on the dimeric hGS enzyme. Mutation of the amino acids identified as the most significant vis-`-vis subunit:subunit communication will be tested using in silico (molecular dynamics) and in vitro site-directed mutagenesis;it is anticipated that mutagenesis of these residues will impact both hGS negative cooperativity and overall enzyme activity. The hypothesis that a loop, specifically the A- loop, of hGS is the conduit by which the subunit active sites communicate, thus leading to negative cooperativity, will be tested. Aim 2: Amino acids in hGS that are in contact with both important dimer interface residues and substrate binding residues will be identified with modeling. To assess whether these residues are involved in negative cooperativity, these amino acids will be mutated in silico and in vitro to understand at an atomic level how such mutations affect the active site geometry of hGS, activity, and negative cooperativity. Human glutathione synthetase is an important enzyme because of the physiological functions of glutathione and also because it serves as a paradigm for other protein families and for allostery. PUBLIC HEALTH RELEVANCE: Human glutathione synthetase (hGS) is an important enzyme because of its role in the synthesis of the physiologically important antioxidant glutathione, and because hGS serves as a model for other protein families. Long-term our research will impact the understanding of allosteric, multimeric and/or ATP- using proteins and also the burgeoning interest in understanding and controlling protein:protein interactions.